Just started to learn Cellprofiler and I am quite enthusiastic about what it can do so far. Starting from the basics, I am simply trying to count cells which are dual positive for two different cell surface markers (e.g. green and red) for now.
Initially, I have set up a pipeline for identify primary object --> identify secondary object. However, this gave me 100% count for my secondary objects. Reading through previous posts here, I realized this was a wrong set up as each primary object corresponds to a secondary object, thus giving identical counts.
I have then progressed to identify primary object (green) --> identify primary object (red) --> Relate object (green as parent, red as child) --> Filter object parent based on child counts, minimum 1, no maximum value. In between each of these steps, I have also adjusted image intensity for each image in green and red channels to reduce background signals.
This is where I ran into problems. By manual counting using other softwares, I believe I am getting at least 90% (and above) dual positive green and red cells. However, CP gives me approximately about 40-60% positive. Unlike previous posts where researchers are looking at nuclei in cells where its fairly clear that nuclei are inside a cell, I am trying to count dual positive cell surface markers. I’m wondering if somehow CP has trouble counting this? Or perhaps I used a wrong pipeline and there might be a better approach to this?
Should I post some example images here (edit: uploaded)?
Thanks for the help and any suggestions!